The cAMP-dependent protein kinase (PKA) is targeted to specific subcellular compartments through its interaction with A kinase anchoring proteins (AKAPs). Membrane permeable peptides, designed to disrupt PKA/AKAP interaction, inhibit sperm motility, suggesting PKA anchoring is required for the maintenance of motility. The overall goal of this proposal is to characterize the interaction of sperm AKAPs with the type two regulatory subunit of PKA (RII) and RII-homologues, and determine how these interactions regulate spermatozoan function(s). We will focus on two sperm AKAPs. AKAP110 is a predominant sperm AKAP and the focus of our previous proposal. Radial Spoke Protein 3 (RSP3) is a newly discovered AKAP in Chlamydomonas. It is the first flagellar AKAP to be found in the axoneme. Mutation of this protein disrupts flagellar beating. We have recently identified, cloned and expressed the human homolog of this protein. Based on the location of this AKAP, we hypothesize that RSP3 regulates motility by coordinating the action of kinases and/or phosphatases in the axoneme. We have recently identified four sperm-specific human proteins, in addition to PKA, which interact with sperm AKAP110. All of these proteins have a strong sequence similarity to the AKAP docking and dimerization domains of RII. Although these sperm proteins appear to be functional homologues of RII in their ability to bind AKAPs, they do not share other functions of RII such as the ability to bind cAMP or the catalytic subunit of PKA. Two of these RII homologues appear to be part of the Rho signaling pathway. One is homologous to murine ropporin. Ropporin is found along the principal piece of the flagellum and binds to rhophilin, a target protein for Rho. A second protein, named AKAP-associated sperm protein (ASP), is 39 percent identical with ropporin. Agents that inhibit Rho signaling inhibit sperm motility. One goal of this proposal is to test the hypothesis that Rho signaling regulates sperm motility via a mechanism similar to that observed in smooth muscle and that sperm AKAPs coordinate this mechanism by acting as scaffolding molecules. Studies outlined in this proposal should facilitate the design of new and highly specific pharmacological reagents for inhibiting sperm function in vivo. Knowledge gained from these studies may also be useful for the diagnosis and treatment of patients with defects in sperm movement and fertility.